The invention is directed to new derivatives of the pentapeptide, splenopentin, arg-lys-glu-val-tyr, methods for their synthesis and purification, as well their use as immunobiologically active pharmaceutical preparations.
According to the present invention, splenopentin and its derivatives are used as immunological regulators in medical therapy. The field of application of the invention is the pharmaceutical industry.
As a result of extensive investigations of the isolation and characterization of peptides, which affect the immunological system, such materials as the thymopoietins and splenin (T, Audhya et al., Biochemistry 20, 6195 (1981)) have been isolated from the thymus or the spleen in recent years.
Although they originate from different organs, these peptides largely have a structural similarity. They consist of 49 amino acids and their sequence differs only in position 34 due to aspartic or glutamic acid. On the other hand, they differ clearly in their biological activity. For example, thymopoietins affect neuromuscular transmission (M.P. Scheid et al., J. Exp. med. 147, 1727 (1978)), stimulate the differentiation of early T cells and inhibit the differentiation of early B cells.
On the other hand, splenin stimulates the differentiation of T and B cells and has no neuroactivity. The biological activity of the splenin molecule as a whole is reproduced by its partial sequence 32-36, arg-lys-glu-val-tyr (splenopentin).
The synthesis of biologically active substances assumes to a constantly greater extent the simultaneous development of more highly sensitive and economically more effective chromatographic methods of purification. Of prime importance in this connection is the use of those methods, which have a high substance throughput, a uniformly good recovery rate and a high system resolution, when simple support and solvent systems are used.
Because of the slight charge and polarity differences of a large number of components, the chromatographic separation of differently acylated splenopentin peptides requires the use of more expensive and costly chromatographic techniques. Preferably, HPLC methods, based on reverse-phase silica gels, as well as method of polar adsorption and partition chromatography on normal silica gel phases are used.
According to the proposal of the DE 3 421 614 (1984), the chromatographic purification of splenopentins is carried out on normal-phase silica gel in methylene chloride-containing solvents, Furthermore, the proposal has already been made that the chromatographic separation of crude, synthetic splenopentin products be carried out by adsorption or partition chromatography on silica gel 60, using pyridine-containing organic solvents. Similar methods of separation are proposed in DE 2 804 566 (1977) for the purification of thymopentin or monoacetyl thymopentin.
It is a disadvantage of methods based on reverse-phase or normal-phase silica gel that the cost of the equipment and the material is high. This precludes their use for the production of large amounts of products of high purity. Moreover, the low capacity of the system, the low recovery rates due to nonspecific adsorption, as well as its inadequate ability to separate, are limiting factors for its application.
The usual ion exchange/chromatographic purification methods for peptides are characterized by the use of salts during the elution process. The chromatographic purification of nona- and decapeptides, described in DE 2,732,587 as well as in DE 2,617,646 is accomplished by the use of carboxymethylcellulose with elution agents containing sodium chloride or ammonium acetate. For purifying the substances, an expensive desalinization is therefore subsequently always necessary.
It is an object of the invention to develop new immunologically active derivatives of splenopentin (SP5) and methods for their synthesis and purification and to make immunologically effective pharmaceutical preparations available.